Methods and Compositions for Increasing the Amounts of Phosphorus Available for Plant Uptake from Soils

ABSTRACT

The present invention relates to a method of enhancing growth conditions for plants by growing the plants in soil containing, in proximity to the plant roots, both a phosphorus source and at least two strains of the fungus  Penicillium , particularly  P. bilaiae , more particularly strains NRRL 50162 and NRRL 50169.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/791,721 filed on Jul. 6, 2015 (now allowed), which is a continuationof U.S. application Ser. No. 13/595,367 filed on Aug. 27, 2012 (now U.S.Pat. No. 9,101,088), which is a continuation of U.S. application Ser.No. 12/571,913 filed Oct. 1, 2009 (now U.S. Pat. No. 8,278,247), whichclaims priority or the benefit under 35 U.S.C. 119 of European patentapplication no. EP 08 165 591.2 filed Oct. 1, 2008 and U.S. provisionalapplication No. 61/102,603 filed Oct. 3, 2008, the contents of which arefully incorporated herein by reference.

REFERENCE TO A DEPOSIT OF BIOLOGICAL MATERIAL

This application contains a reference to deposits of biologicalmaterial, which deposits are incorporated herein by reference. Forcomplete information see last 2 pages of the description.

FIELD OF THE INVENTION

The present invention relates to a method of increasing the availabilityof phosphorus for plant uptake from soil, to a composition forapplication to soil and to a plant seed.

BACKGROUND OF THE INVENTION

In order to maintain healthy growth, plants must extract a variety ofelements from the soil in which they grow. These elements includephosphorus and the so-called micro-nutrients (e.g. copper, iron andzinc), but many soils are deficient in such elements or they containthem only in forms which cannot be readily taken up by plants (it isgenerally believed that essential elements cannot be readily taken up byplants unless they are present in dissolved form in the soil).

To counteract such deficiencies, sources of the deficient elements arecommonly applied to soils in order to improve growth rates and yieldsobtained from crop plants. For example, phosphates are often added tosoil to counteract a lack of available phosphorus. Phosphate added tothe soil as a commercial fertilizer (e.g., mono-ammonium phosphate ortriple-super-phosphate) is readily plant available, but is rapidlyconverted in soil to relatively unavailable forms. It has been estimatedthat only 10 to 30% of phosphate fertilizer is used by the plant in theyear it is applied, and one-third to one-half of the phosphatefertilizer applied may never be recovered by the plant.

Attempts have been made in the past to use microorganisms to improve theavailability of essential elements in soil systems. In particularspecies of the fungus Penicillium has been used for this purpose. U.S.Pat. No. 5,026,417 describes an isolated strain of P. bilaiae which iscapable of improving the uptake of phosphorous by plants when applied tothe soil.

There is, however, still a need for systems for improving growthconditions for plants, particularly by increasing the levels ofavailable phosphorus in soil systems.

SUMMARY OF THE INVENTION

The present invention is based on the finding that different species ofPenicillium, which when applied alone may have varying ability toimprove the availability of phosphorus both from insoluble phosphatesand from manufactured fertilizers, can when combined give rise to asynergistic effect that surpasses what could be expected from individualresults.

In particular this holds true for Penicillium species belonging to P.bilaiae.

The invention provides in a first aspect a method of increasing theavailability of phosphorus for plant uptake from soil, which methodcomprises introducing into the soil inoculums of at least two differentstrains of the fungus Penicillium.

In a second aspect the invention relates to a method of enhancing growthconditions of plants, which comprises growing the plants in soilcontaining, in proximity to the plant roots, both a phosphorus sourceand at least two strains identified by the deposit numbers NRRL 50169and NRRL 50162.

In a third aspect the invention relates to a composition for applicationto soil, which comprises: i) inoculums of at least two strains of thefungus Penicillium, particularly P. bilaiae and/or P. gaestrivorus, andii) a soil-compatible carrier for the fungus.

In a forth aspect the invention relates to a plant seed having a coatingcomprising inoculums of at least two strains of the fungus Penicillium,in particular P. bilaiae and/or P. gaestrivorus, and a solidsoil-compatible carrier therefore.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the effect of inoculation with phosphate solubilisingmicro-organisms on yield of corn. P. bilaiae (Novozymes P-201)corresponds to strain NRRL 50169, and P. bilaiae (Australia, P-208)corresponds to NRRL 50162.

FIG. 2 shows hydroxyl apatite solubilisation in liquid culture by P.bilaiae strains singly and in combination. Numbers in brackets indicateincubation rate (spores per flask).

FIG. 3 shows the effect of inoculation with phosphate solubilisingmicroorganisms on the shoot dry weight of soybean grown under growthroom conditions. P. bilaiae (Novozymes) corresponds to strain NRRL50169, P. bilaiae (Australia) corresponds to NRRL 50162, and P. bilaiaeblend corresponds to a 1:1 blend of the two strains.

FIG. 4 shows the effect of inoculation with phosphate solubilizingmicroorganisms on shoot dry weight of corn grown under growth roomconditions. P. bilaiae (Novozymes) corresponds to strain NRRL 50169, P.bilaiae (Australia) corresponds to NRRL 50162, and P. bilaiae blendcorresponds to a 1:1 blend of the two strains.

DETAILED DESCRIPTION OF THE INVENTION

The fungus Penicillium bilaiae is a known micro-organism that haspreviously been deposited at the American Type Culture Collection inRockville, Md., USA under the deposit number ATCC 22348 (1974 edition ofthe ATCC catalogue). In the 1984 catalogue, the same deposit number isused for P. bilaii and a further strain is identified by the depositnumber 18309.

Further isolates of this fungus has been discovered in soil from alocation (latitude 49.degree. 48′ N, longitude 113.degree. 6′ W) inSouthern Alberta, Canada. This strain has previously been shown toimproved P-solubilizing activity compared to the earlier strainsdeposited at the ATCC. A deposit of this improved strain was made at theATCC under the deposit number 20851 in accordance with the terms of theBudapest Treaty. In this deposit the fungus was named P. bilaji and thetaxonomic details and it use has been described in U.S. Pat. No.5,026,417. This strain has now been re-deposited as NRRL 50169. Forcomplete information of the deposit see last page of the description.

The name of this species has subsequently been changed again and is nowrecognized as P. bilaiae. This name will consequently be used throughoutthe specification.

A new isolate of P. bilaiae has been discovered in Australia. It wasoriginally isolated in 2002 from wheat roots and grown in collected soilsamples from Coonalpyn in South Australia (Wakelin et al., 2004. BiolFertil Soils 40:36-43). A deposit of this improved strain was made asdeposit number NRRL 50162. For complete information of the deposit seelast page of the description and the taxonomic details of this isolateand its proposed use is described in US provisional application filed on1 Oct. 2008 in the name of CSIRO.

Other Penicilium spp. found to be particularly useful according to thepresent invention are strains of P. gaestrivorus. One such strain wasisolated in 2002 from wheat roots grown in collected soil samples fromNew South Wales, Australia (Wakelin et al., 2004. Biol Fertil Soils40:36-43), and deposited as NRRL 50170. For complete information of thedeposit see last page of the description.

According to one aspect the invention relates to a method of enhancinggrowth conditions of plants, comprising growing the plants in soilcontaining, in proximity to the plant roots, both a phosphorus sourceand at least two strains of the fungus Penicillium. Particularly thePenicilium fungus is selected from P. bilaiae and/or P. gaestrivorus. Inparticular the enhanced growth is provided by enhancing the availabilityof phosphorus for plant uptake from soil. In a particular embodiment thePenicillium strains are selected from the strain deposited as NRRL 50169and NRRL 50162.

The use of a combination of at least two different Penicillium strainshas the following advantages. When applied to soil already containinginsoluble (or sparingly soluble) phosphates, the use of the combinedfungal strains will result in an increase in the amount of phosphorusavailable for plant uptake compared to the use of only one Penicilliumstrain. This in turn may result in an increase in phosphate uptakeand/or an increase in yield of plants grown in the soil compared to useof individual strains alone. If e.g. phosphorous is not a limitingfactor a yield increase may not necessarily follow as a result of theincreased availability. The combination of strains also enablesinsoluble rock phosphates to be used as an effective fertilizer forsoils which have inadequate amounts of available phosphorus.

According to one aspect the invention therefore relates to a method ofincreasing the availability of phosphorus for plant uptake from soil,which method comprises introducing into the soil inoculums of at leasttwo different strains of the fungus Penicillium. The presence of the twostrains of Penicillium will enhance the availability of phosphorus forplant uptake.

The said phosphorus may be provided from a source selected from thegroup consisting of sources originally present in the soil, sourcesadded to the soil as amendments and combinations thereof.

The term “inoculum” as used in this specification is intended to meanany form of fungus cells, mycelium or spores, which is capable ofpropagating on or in the soil when the conditions of temperature,moisture, etc., are favorable for fungal growth.

By “source” of a particular element we mean a compound of that elementwhich, at least in the soil conditions under consideration, does notmake the element fully available for plant uptake.

In particular the Penicillium fungus is selected from the groupconsisting of sp. P. bilaiae, P. albidum, P. aurantiogriseum, P.chrysogenum, P. citreonigrum, P. citrinum, P. digitatum, P. frequentas,P. fuscum, P. gaestrivorus, P. glabrum, P. griseofulvum, P. implicatum,P. janthinellum, P. lilacinum, P. minioluteum, P. montanense, P.nigricans, P. oxalicum, P. pinetorum, P. pinophilum, P. purpurogenum, P.radicans, P. radicum, P. raistrickii, P. rugulosum, P. simplicissimum,P. solitum, P. variabile, P. velutinum, P. viridicatum, P. glaucum, P.fussiporus, and P. expansum.

In one particular embodiment the Penicillium species is P. bilaiae. Inanother particular embodiment the Penicillium species is P.gaestrivorus. In a further particular embodiment the at least twostrains are one strain of P. bilaiae and one strain of P. gaestrivorus.

In another particular embodiment the P. bilaiae strains are selectedfrom the group consisting of ATCC 20851, NRRL 50169, ATCC 22348, ATCC18309, NRRL 50162.

In an even further embodiment the at least two strains are NRRL 50169and NRRL 50162.

In a still further embodiment the at least two strains are NRRL 50169and NRRL 50170.

In a still further embodiment the at least two strains are NRRL 50162and NRRL 50170.

The Penicillium fungus according to the invention and in particular thespecific strains, ATCC20851, NRRL 50169, NRRL 50170 and NRRL 50162 canbe grown using solid state or liquid fermentation and a suitable carbonsource. Penicillium isolates may be grown using any suitable methodknown to the person skilled in the art. For example, the fungus may becultured on a solid growth medium such as potato dextrose agar or maltextract agar, or in flasks containing suitable liquid media such asCzapek-Dox medium or potato dextrose broth. These culture methods may beused in the preparation of an inoculum of Penicillium spp. for coatingseeds and/or application to carrier to be applied to soil.

Solid state production of Penicillium spores may be achieved byinoculating a solid medium such as a peat or vermiculite-basedsubstrate, or grains including, but not limited to, oats, wheat, barley,or rice. The sterilized medium (achieved through autoclaving orirradiation) is inoculated with a spore suspension (1×10²-1×10⁷ cfu/ml)of the appropriate Penicillium spp. and the moisture adjusted to 20 to50%, depending on the substrate. The material is incubated for 2 to 8weeks at room temperature. The spores may also be produced by liquidfermentation (Cunningham et al., 1990. Can J Bot 68:2270-2274). Liquidproduction may be achieved by cultivating the fungus in any suitablemedia, such as potato dextrose broth or sucrose yeast extract media,under appropriate pH and temperature conditions (as could be performedby anyone skilled in the art).

The resulting material may be used directly as a seed treatment, or thespores may be harvested, concentrated by centrifugation, formulated, andthen dried using air drying, freeze drying, or fluid bed dryingtechniques (Friesen T., Hill G., Pugsley T., Holloway G., and ZimmermanD. 2005, Experimental determination of viability loss of Penicilliumbilaiae conidia during convective air-drying Appl Microbiol Biotechnol68: 397-404) to produce a wettable powder. The wettable powder is thensuspended in water, applied to the surface of seeds, and allowed to dryprior to planting. The wettable powder may be used in conjunction withother seed treatments, such as, but not limited to, chemical seedtreatments, carriers (e.g., talc, clay, kaolin, silica gel, kaolinite)or polymers (e.g., methylcellulose, polyvinylpyrrolidone).Alternatively, a spore suspension of the appropriate Penicillium spp.may be applied to a suitable soil-compatible carrier (e.g., peat-basedpowder or granule) to appropriate final moisture content. The materialis incubated at room temperature for 2 to 8 weeks, and can then beapplied to the soil in the furrow along with the seed.

As described above, it has been found that the combination of at leasttwo strains of Penicillium increases the amount of phosphorus availablefor plant uptake from commercial phosphorus fertilizers compared to theuse of only one strain so commercial fertilizers may be added to thesoil instead of (or even as well as) natural rock phosphate.

According to further embodiments of the invention the source ofphosphorous comprises a source of phosphorous native to the soil or inanother embodiment the source of phosphorous is added to the soil.

In one embodiment said source is rock phosphate. In another embodimentsaid source is a manufactured fertilizer.

Commercially available manufactured phosphate fertilizers are of manytypes. Some common ones are those containing monoammonium phosphate(MAP), triple super phosphate (TSP), diammonium phosphate, ordinarysuperphosphate and ammonium polyphosphate. All of these fertilizers areproduced by chemical processing of insoluble natural rock phosphates inlarge scale fertilizer-manufacturing facilities and the product isexpensive. By means of the present invention it is possible to reducethe amount of these fertilizers applied to the soil while stillmaintaining the same amount of phosphorus uptake from the soil.

In a further particular embodiment the source or phosphorus is organic.An organic fertilizer refers to a soil amendment derived from naturalsources that guarantees, at least, the minimum percentages of nitrogen,phosphate, and potash. Examples include plant and animal by-products,rock powders, seaweed, inoculants, and conditioners. These are oftenavailable at garden centers and through horticultural supply companies.In particular said organic source of phosphorus is from bone meal, meatmeal, animal manure, compost, sewage sludge, or guano.

Other fertilizers, such as nitrogen sources, or other soil amendmentsmay of course also be added to the soil at approximately the same timeas the Penicillium fungus or at other times, so long as the othermaterials are not toxic to the fungus.

Since the fungus has the effect of solubilizing phosphates which mayalready be present in soil (i.e., those which are native to the soil)and also those which are added to the soil, the fungus may be appliedalone to soils which contain native sources of phosphorus, or may beapplied to any soils in conjunction with added sources of phosphorus.The inoculums comprising the fungal strains according to the inventioncan as described above be provided using solid state or liquidfermentation and a suitable carbon source.

The amount of the inoculum to be applied to the soil is not limited inany particular respect. Clearly, if an insufficient amount is used, anoticeable effect will not be obtained. On the other hand, the use oflarge amounts of the inoculum will be wasteful because the amounts ofphosphorus and/or micronutrients made available in the soil reach amaximum at a certain application rate and further additions beyond thisrate do not give additional benefits. The suitable application ratesvary according to the type of soil, the type of crop plants, the amountsof the source of phosphorus and/or micronutrients present in the soil oradded thereto, etc. and a suitable rate can be found without difficultyby simple trial and error experiments for each particular case.Normally, the application rate falls into the range of 0.001-1.0 Kgfungal spores and mycelium (fresh weight) per hectare, or 10²-10⁶ colonyforming units (cfu) per seed (when coated seeds are used), or on agranular carrier applying between 1×10⁶ and 1×10¹¹ colony forming unitsper hectare. Even though the inoculums used according to the presentinvention is comprised of a mixture/combination of inoculums of at leasttwo different strains of Penicillium it is the total amount of spores orcolony forming units in the combined mixture that is referred tothroughout the specification.

The fungal cells in the form of e.g. spores and optionally a carrier canbe added to a seed row of the soil at the root level or can be used tocoat seeds prior to planting. When spores are added to the soil agranular formulation will be preferable. Formulations as liquid, peat,or wettable powder will be suitable for coating of seeds. When used tocoat seeds, the material can be mixed with water, applied to the seedsand allowed to dry.

Other carriers for the spores can be used to coat seeds. For example,the spores can be grown on moistened bran, dried, sieved and applied toseeds prior coated with an adhesive, e.g. gum arabic.

The carrier should preferably be a soil compatible carrier. The term“soil-compatible” means any material which can be added to the soilwithout having an adverse effect on plant growth, soil structure, soildrainage or the like. Suitable carriers comprise, but are not limitedto, wheat chaff, bran, ground wheat straw, peat-based powders orgranules, gypsum-based granules, and clays (e.g., kaolin, bentonite,montmorillonite).

In a further aspect the present invention relates to a compositioncomprising at least two strains of the Penicilium fungus according tothe invention, and a carrier. Suitable carriers include water, aqueoussolutions, slurries, solids (e.g. peat, wheat, bran, vermiculite, andpasteurized soil) or dry powders.

The composition according to the invention may suitably be applied inthe method of the invention for increasing the availability ofphosphorous for plant uptake from soil.

In a particular embodiment the at least two strains of Peniciliumcomprised in the composition are selected from the group consisting ofPenicilium bilaiae and Penicilium gaestrivorus. More particularly thePenicilium strains are selected from the group consisting of NRRL 50169,NRRL 50162, NRRL 50170. In a further specific embodiment the two strainsare NRRL 50169 and NRRL 50162. In another embodiment the two strains areNRRL 50162 and NRRL 50170.

Particularly the carrier may in one embodiment comprise a liquidcontaining a nutrient for the fungus.

In a still further embodiment the present invention relates to a plantseed having a coating comprising inoculums of at least two strains ofthe fungus Penicillium, in particular P. bilaiae and/or P. gaestrivorus,and a solid soil-compatible carrier therefore. More particularly thePenicillium strains are selected from the group consisting of NRRL50169, NRRL 50162, NRRL 50170. In a further specific embodiment the twostrains are NRRL 50169 and NRRL 50162. In another embodiment the twostrains are NRRL 50162 and NRRL 50170.

The composition may contain additional additives including bufferingagents, wetting agents, coating agents, and abrading agents.

The methods according to the invention are potentially useful forimproving growth conditions resulting in increased phosphorous uptakeand/or yield for any type of plant. In one particular embodiment theplant is selected from the group consisting of cereals, legumes,Brassica spp., fruits, vegetables, nuts, flowers, and turf. Particularlythe cereals are wheat, corn, rice, oat, rye, barley. Particularlylegumes are lentil, chickpeas, beans, soybeans, peas, and alfalfa.

In another particular embodiment the plants are selected from the groupconsisting of alfalfa, rice, wheat, barley, rye, oat, cotton, sunflower,peanut, corn, potato, sweet potato, bean, pea, chickpeas, lentil,chicory, lettuce, endive, cabbage, brussel sprout, beet, parsnip,turnip, cauliflower, broccoli, turnip, radish, spinach, onion, garlic,eggplant, pepper, celery, carrot, squash, pumpkin, zucchini, cucumber,apple, pear, melon, citrus, strawberry, grape, raspberry, pineapple,soybean, tobacco, tomato, sorghum, and sugarcane.

EXAMPLES Example 1 Characterization of Isolates

Genetic analysis of the D2 region of 28S rDNA has confirmed the twostrains ATCC 20851 (P-201 strain; same as NRRL 50169) and NRRL 50162(P-208 strain) as being P. bilaiae, with additional work at CSIRO(Australia) proving them to be different strains. Strains of P. bilaiaewere sequenced by MIDI Labs in Newark, Del. using universal primers tothe D2 region of the 28S rDNA gene. Phylogenetic comparison calculationswere done using the program CLUSTAX to align the sequence to otherclosely related species indicated by an initial BLAST analysis of thesequence. Once the multiple alignment file was created, a NeighborJoining tree was constructed, and a distance matrix was calculated asthe basis for identifying the genus and species of the strain. Allalignment and phylogenetics related operations were done in Mega 4.0.Sequences were imported into the Alignment Explorer in Mega, and thenaligned using ClustalW. A phylogenetic tree was constructed usingbootstrapping to test the robustness.

Identifications of the two strains were confirmed as Penicillium bilaiaeaccording to the following classification:

Kingdom Fungi Subkingdom Dikarya Phylum Ascomycota SubphylumPezizomycotina Class Eurotiomycetes Subclass Eurotiomycetidae OrderEurotiales Family Trichocomaceae Subfamily mitosporic (anamorphic)Trichocomaceae Genus Penicillium Species bilaiae

Example 2 Field Trials of Combination Treatment

Field trials were established in 2007 at four USA locations to screenthe effect of inoculation with phosphate solubilizing strains of P.bilaiae on the harvested yield of corn. Treatments included two strainsof P. bilaiae singly and in combination as well as an uninoculatedcontrol.

The trials were established at four USA locations as randomized completeblocks with 6 replicates per trial. The USA trials were conducted byfour independent research firms covering four USA States. The researchcontractors and locations were Viger Ag Research (Fergus Falls, Minn.),Benson Research (York, Nebr.), Northern Plains Ag (Gardner, N. Dak.),and South Dakota Ag Research (Centerville, S. Dak.).

The trial was composed of four treatments that included two singlestrain Penicillium treatments NRRL 50169 (Novozymes P-201 strain) andNRRL 50162 (Australian P-208 strain), a double strain treatment, and anun-inoculated control. All Penicillium strains were formulated as peatgranules.

Production of peat granules was achieved by inoculating the substratewith a liquid spore suspension. Cultures of Penicillium were taken fromstorage at −80° C. and grown on potato dextrose agar. Spores werecollected by using a glass rod to scrape the surface of a sporulatingculture (obtained after two weeks of incubation at room temperature)into sterile water amended with 0.1% v/v Tween 80. The spore suspensionof the appropriate Penicillium spp. was added to peat granules, whichwere then mixed thoroughly to achieve a uniform inoculation, stored inplastic-lined paper bags, and incubated for 2 to 8 weeks at roomtemperature (approximately 22 to 27° C.). Bags were randomly sampled andanalyzed to approximate the fungal colony forming units for each lot ofinoculated granules. Briefly, a dilution series was made using sterilewater amended with 0.1% v/v Tween 80. Aliquots of the dilutions wereplated on potato dextrose agar supplemented with Rose Bengal andchlorotetracycline. Fungal colonies were counted after 3 to 5 daysincubation at approximately 25° C. For combination treatments,inoculated granules were blended to achieve a 1:1 blend of the P.bilaiae isolates by titre.

Field plot establishment was specific to each site (see Table 1). Seedrow spacing was 30 inches, with 2 rows of corn per plot plus two guardrows. Fertilization included a standard nitrogen fertility program (sitespecific), plus 10 kg ha⁻¹ P₂O₅ applied with the seed. Penicilliumstrains were formulated as peat granules and applied in furrow at a rateof 4.5 kg ha⁻¹ (2.07 to 2.17E+10 colony forming units ha⁻¹).

TABLE 1 Details of field plot establishment at four USA locations. Seedsize (g 100 Seeding rate Site Seed variety kernels⁻¹) (seeds ac⁻¹) Plotsize Fergus Falls, DK 40-07 21.25 34,000 10 × 20 ft MN York, NECornhusker 28.33 33,000 525 ft² Hybrids 1 Gardner, ND Mycogen 32.6632,000 7.3 × 30 ft 2K154 Centerville, NK 51-T8 29.46 28,000 10 × 30 ftSD

Combined trial analysis shows significantly higher corn yield in the P.bilaiae combination treatment compared to the uninoculated control (FIG.1). The P. bilaiae combination out-yielded either of the P. bilaiaestrains when used as a separate treatment. The P. bilaiae straincombination was the most impactful treatment on corn yield in thesestudies.

Example 3 Solubilization of Insoluble Phosphates

Phosphate solubilizing strains of P. bilaiae have been included inlaboratory experiments examining the ability of these organisms tosolubilize insoluble calcium phosphates. The experiment consisted of twostrains of P. bilaiae singly and in combination at two differentconcentrations, as well as an uninoculated control.

Hydroxyapatite was weighed into 300-mL Erlenmeyer flasks at a rate of100 mg P per flask. Minimal salts media was prepared as follows (g L⁻¹):0.1 NaCl, 0.4 NH₄Cl, 0.78 KNO₃, 0.1 CaCl₂.2H₂O, 1.0 MgSO₄.7H₂O, 10.0sucrose. 100 mL of media was added to each flask, and flasks wereplugged with a foam stopper and autoclaved (121° C. and 1.2 atm for 30min). Triplicate flasks were inoculated with liquid spore stocks to atarget inoculation rate of 4.00E+06 spores per flask. For the P. bilaiaecombination treatments, the flasks were inoculated either with 4.00E+06spores of each strain per flask, or to a total spore concentration of4.00E+06 spores per flask (i.e., 2.00E+06 spores per flask of eachstrain). Flasks were incubated at ambient room temperature on a rotaryshaker set to 175 rpm. Subsamples were aseptically collected at 3, 5, 7,and 10 days after inoculation and analyzed for soluble phosphate using amalachite-green method.

The combination of P. bilaiae strains was able to solubilizesignificantly more hydroxyapatite than either strain alone, as indicatedby increased soluble phosphate levels, with rate of inoculation havelittle to no effect (FIG. 2).

Example 4 Growth Room Trials of Combination Treatment

Growth room trials were established in 2009 to screen the effect ofinoculation with phosphate solubilizing strains of P. bilaiae on the drymatter accumulation of soybean and corn. Treatments included two strainsof P. bilaiae singly and in combination as well as an uninoculatedcontrol.

The trials were established as randomized factorials with 8 replicatesper trial. The two factors were phosphate fertilizer and inoculant.Fertilizer levels were equivalent to 0, 20, 40, and 80 lb P₂O₅ ac⁻¹ forsoybean, and 0, 40, 80, and 160 lb P₂O₅ ac⁻¹ for corn. Inoculanttreatments were composed of an uninoculated control, two single strainPenicillium treatments [NRRL 50169 (Novozymes P-201 strain) and NRRL50162 (Australian P-208 strain)], and a double-strain treatmentinvolving equal amounts of both Penicillium strains.

Plastic pots were labeled according to treatment and a sterile square ofblack landscape fabric was placed in the bottom of each to prevent thepotting mix from leaking through the drainage holes. Pots were filledwith a 1:1 mixture of industrial quartz sand and fine-milledvermiculite. Each pot was treated with the appropriate rate of phosphatesuspension prepared using hydroxyapatite, sealed inside a Ziploc bag,and allowed to equilibrate for 7 days prior to seeding.

Inoculant treatments were applied as a liquid seed treatment. Culturesof Penicillium were taken from storage at −80° C. and grown on potatodextrose agar. Spores were collected by using a glass rod to scrape thesurface of a sporulating culture (obtained after two weeks of incubationat room temperature) into sterile water amended with 0.1% v/v Tween 80.The spore suspensions were titred, mixed thoroughly, and added to seedlots pre-weighed into plastic bags at a rate of 1.50E+05 colony formingunits per seed. For combination treatments, a 1:1 blend of P. bilaiaeisolates was achieved by halving the volume of spore suspension requiredto reach the target inoculation rate for each strain (i.e., finalinoculation rate remained 1.50E+05). Uninoculated control treatmentswere treated with sterile water. The plastic bags were sealed and shakenvigorously for 1 to 2 min to evenly coat the seeds. Bags were re-opened,and the seeds were allowed to dry for 20 to 30 min prior to planting.

Soybean and corn seeds were planted 5 per pot. Phosphate-free nutrientsolution was added to pots at the time of seeding, and every two weeksfor the duration of the experiment. Pots were placed into the growthroom with day/night settings of 16/8 h and 20/15° C., and watered on asrequired. Pots were thinned to three seedlings one to two weeks afterplanting. Plants were harvested approximately 7 and 6 weeks afterplanting for soybean and corn, respectively. Shoots were removed abovethe soil line, placed inside pre-weighed paper bags, and dried for 10 dat 72° C. to determine the dry shoot weight.

Across all fertilizer levels, soybean showed higher shoot dry matteraccumulation in the P. bilaiae blend treatment compared to theuninoculated control (FIG. 3). The P. bilaiae blend treatment alsoperformed better than either of the P. bilaiae strains alone.

Corn showed higher shoot dry matter accumulation in the P. bilaiae blendtreatment than the uninoculated control for all fertilizer levels (FIG.4). The P. bilaiae combination treatment also performed better thaneither of the P. bilaiae strains alone at the 40 and 80 lb P₂O₅ ac⁻¹fertilizer rates.

Deposit of Biological Material

The following biological material has been deposited under the terms ofthe Budapest Treaty with the Agricultural Research Service PatentCulture Collection (NRRL), Northern Regional Research Center, 1815 N.University Street, Peoria, Ill., 61604, USA, and given the followingaccession number:

Deposit Accession Number Date of Deposit Penicillium bilaiae NRRL 50169Aug. 28, 2008

The following biological material has been deposited under the terms ofthe Budapest Treaty with the Agricultural Research Service PatentCulture Collection (NRRL), Northern Regional Research Center, 1815 N.University Street, Peoria, Ill., 61604, USA, and given the followingaccession number:

Deposit Accession Number Date of Deposit Penicillium bilaiae NRRL 50162Aug. 11, 2008

The following biological material has been deposited under the terms ofthe Budapest Treaty with Agricultural Research Service Patent CultureCollection (NRRL), Northern Regional Research Center, 1815 N. UniversityStreet, Peoria, Ill., 61604, USA and given the following accessionnumber:

Deposit Accession Number Date of Deposit Penicillium gaestrivorus NRRL50170 Aug. 28, 2008

The strains have been deposited under conditions that assure that accessto the culture will be available during the pendency of this patentapplication to one determined by foreign patent laws to be entitledthereto. The deposit represents a substantially pure culture of thedeposited strain. The deposit is available as required by foreign patentlaws in countries wherein counterparts of the subject application, orits progeny are filed. However, it should be understood that theavailability of a deposit does not constitute a license to practice thesubject invention in derogation of patent rights granted by governmentalaction.

1. A method of increasing corn yield, said method comprising introducingPenicillium bilaiae NRRL 50162 and Penicillium bilaiae NRRL 50169 intosoil in which corn will be grown, wherein the introduction ofPenicillium bilaiae NRRL 50162 and Penicillium bilaiae NRRL 50169 intosaid soil synergistically increases the yield of corn grown in saidsoil.
 2. The method of claim 1, wherein said Penicillium bilaiae NRRL50162 is introduced into said soil in an amount that is insufficient toincrease the yield of corn grown in said soil as compared to corn grownin untreated soil.
 3. The method of claim 1, wherein said Penicilliumbilaiae NRRL 50169 is introduced into said soil in an amount that isinsufficient to increase the yield of corn grown in said soil ascompared to corn grown in untreated soil.
 4. The method of claim 1,wherein said Penicillium bilaiae NRRL 50162 is introduced into said soilin an amount that is insufficient to increase the yield of corn grown insaid soil as compared to corn grown in untreated soil and wherein saidPenicillium bilaiae NRRL 50169 is introduced into said soil in an amountthat is insufficient to increase the yield of corn grown in said soil ascompared to corn grown in untreated soil.
 5. The method of claim 1,wherein Penicillium bilaiae NRRL 50162 and Penicillium bilaiae NRRL50169 are introduced into said soil as part of a composition comprisingPenicillium bilaiae NRRL 50162, Penicillium bilaiae NRRL 50169 and apeat-based powder or granule.
 6. The method of claim 1, whereinPenicillium bilaiae NRRL 50162 and Penicillium bilaiae NRRL 50169 areintroduced into said soil as part of a composition comprisingPenicillium bilaiae NRRL 50162, Penicillium bilaiae NRRL 50169 and aliquid carrier.
 7. The method of claim 1, wherein Penicillium bilaiaeNRRL 50162 and Penicillium bilaiae NRRL 50169 are introduced into saidsoil as part of a seed that is coated with a composition comprisingPenicillium bilaiae NRRL 50162 and Penicillium bilaiae NRRL
 50169. 8.The method of claim 1, wherein Penicillium bilaiae NRRL 50162 andPenicillium bilaiae NRRL 50169 are introduced into said soil in anamount ranging from 1×10⁶ to 1×10¹¹ colony forming units per hectare. 9.The method of claim 1, further comprising introducing a source ofphosphorous into said soil.
 10. A composition for increasing corn yield,said composition comprising Penicillium bilaiae NRRL 50162, Penicilliumbilaiae NRRL 50169 and a carrier, wherein Penicillium bilaiae NRRL 50162and Penicillium bilaiae NRRL 50169 are present in said composition inamounts sufficient to cause a synergistic yield increase when saidcomposition is introduced into soil in which corn will be grown.
 11. Thecomposition of claim 10, wherein Penicillium bilaiae NRRL 50162 ispresent in said composition in an amount that is insufficient toincrease the yield of corn grown in said soil as compared to corn grownin untreated soil.
 12. The composition of claim 10, wherein saidPenicillium bilaiae NRRL 50169 is present in said composition in anamount that is insufficient to increase the yield of corn grown in saidsoil as compared to corn grown in untreated soil.
 13. The composition ofclaim 10, wherein said Penicillium bilaiae NRRL 50162 is present in saidcomposition in an amount that is insufficient to increase the yield ofcorn grown in said soil as compared to corn grown in untreated soil andwherein said Penicillium bilaiae NRRL 50169 is present in saidcomposition in an amount that is insufficient to increase the yield ofcorn grown in said soil as compared to corn grown in untreated soil. 14.The composition of claim 10, wherein said carrier is a peat-based powderor granule.
 15. The composition of claim 10, wherein said carrier is aliquid carrier.
 16. A seed coated with the composition of claim
 10. 17.The seed of claim 16, wherein Penicillium bilaiae NRRL 50162 andPenicillium bilaiae NRRL 50169 are present on said seed in an amountranging from 1×10¹ to 1×10⁸ colony forming units per seed.
 18. The seedof claim 16, wherein Penicillium bilaiae NRRL 50162 and Penicilliumbilaiae NRRL 50169 are present on said seed in an amount ranging from1×10² to 1×10⁶ colony forming units per seed.
 19. A method comprisingintroducing the composition of claim 10 into a soil.
 20. A methodcomprising planting the seed of claim 16 in a soil.